CN108192597A - Half cyanines class fluorescence probe of near-infrared for detecting butyrylcholine esterase and its preparation method and application - Google Patents
Half cyanines class fluorescence probe of near-infrared for detecting butyrylcholine esterase and its preparation method and application Download PDFInfo
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- CN108192597A CN108192597A CN201611121637.1A CN201611121637A CN108192597A CN 108192597 A CN108192597 A CN 108192597A CN 201611121637 A CN201611121637 A CN 201611121637A CN 108192597 A CN108192597 A CN 108192597A
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Abstract
The present invention relates to the detection fields of butyrylcholine esterase, it discloses a kind of for detecting half cyanines class fluorescence probe of near-infrared of butyrylcholine esterase and its preparation method and application, specifically, the half cyanines class fluorescence probe of near-infrared has the structure shown in formula (1).The method includes in the presence of necleophilic reaction condition and acid binding agent, in organic solvent, formula (2) compound represented is contacted with Cyclopropyl carbonyl chloride.Using including：Application in the activity of detection butyrylcholine esterase, the detection method and detection kit of a kind of activity of butyrylcholine esterase.The half cyanines class probe compound of near-infrared of the present invention for related with carboxypeptidase y Activity determination in application, high sensitivity, good, fluorescence intensity the variation of selectivity are easy to detect greatly, stability higher, using it is more convenient, price is less expensive.
Description
Technical field
The present invention relates to the detection fields of butyrylcholine esterase, and in particular, to a kind of half cyanines class fluorescence probe of near-infrared,
The preparation method of the half cyanines class fluorescence probe of near-infrared and the application in the activity of detection butyrylcholine esterase and a kind of fourth
The detection method of activity of acetylcholinesterase and the detection kit of the activity of BuChE.
Background technology
Butyrylcholine esterase (BChE) is mainly present in a kind of isodynamic enzyme of acetylcholinesterase in organism, it be by
A kind of nonspecific esterase of liver synthesis, is immediately released into blood plasma after being synthesized in liver due to the enzyme, in serum
BChE activity is to measure the sensitive indexes of Hepatocyte matter complex functionality.With horizontal continuous of detection technique and clinic diagnosis
It improves, butyrylcholine esterase extends also to malignant tumour, nervous system disease, the heart in addition to being used to assess liver reserve function
In the detect and diagnose field of numerous diseases such as vascular diseases, metabolic syndrome.In addition, in clinic, serum choline is measured
Esterase active is also to assist diagnosis organic phosphorus compound (such as organic phostoxin gas, organophosphorus pesticide) and cocaine poisoning is important
Approach.
There are mainly three types of the existing methods for measuring the activity of BuChE.The first is to use ultraviolet absorption spectroscopy (i.e.
Ellman methods) cholinesterase activity in sample to be tested is tested, mainly by with acetylthiocholine or thio butyryl
For choline as substrate, the thiocholine generated generates yellow with Ellman reagents (5,5- bis- thiobis -2- nitrobenzoic acids)
Substance.Second is to test the cholinesterase activity in sample to be tested with fluorescent spectrometry, mainly by with thioacetyl
Choline or thio BuCh carry out the activity of indirect monitoring enzyme as substrate using the fluorescence probe for identifying thiocholine.The
Three kinds of natural substrate acetylcholines or BuCh for directly using isotope labelling, directly measure the cholinesterase in sample to be tested
Activity.
In above method, before two kinds belong to indirect method, be easy to cause error；Although the third method is direct side
Method, but operating process is complex, and the large-scale instrument for being required to costliness could be realized with testing cost, also to causing safely
Very big pressure.Moreover, above-mentioned three kinds of methods cannot distinguish between butyrylcholine esterase and acetylcholinesterase, because being surveyed
The activity obtained is the gross activity of all cholinesterases.
Therefore, it designs and develops based on organic molecule as direct substrate type fluorescence probe, and then establishes sensitive, simple
Single, economic specific the activity of BuChE detection method is very urgent and necessary.However so far, both at home and abroad
It is less to be used to detect butyrylcholine esterase (No. EC about specific substrate type fluorescence probe:3.1.1.8) active report.
Invention content
It is an object of the invention to overcome drawbacks described above of the prior art, provide it is a kind of it is highly selective, highly sensitive,
Property is stable, it is easy to operate, be easy to storage and transportation and low-cost non-peptide substrates type butyrylcholine esterase detection fluorescence probe and its
Preparation method and application.
In order to achieve the above objectives, in a first aspect, the present invention provides a kind of half cyanines class fluorescence probe of near-infrared, feature exists
In the half cyanines class fluorescence probe of near-infrared has the structure shown in formula (1)：
Second aspect, the present invention provides the preparation method of above-mentioned half cyanines class fluorescence probe of near-infrared, this method is included in
In the presence of necleophilic reaction condition and acid binding agent, in organic solvent, structural compounds shown in formula (2) are connect with Cyclopropyl carbonyl chloride
It touches；
The third aspect, the present invention provide above-mentioned half cyanines class fluorescence probe of near-infrared in the activity of detection butyrylcholine esterase
Application.
Fourth aspect, the present invention provides a kind of detection method of the activity of butyrylcholine esterase, wherein, this method packet
It includes：
Sample to be tested is contacted with half cyanines class fluorescence probe of near-infrared as described above so that the butyryl courage in sample to be tested
Alkali esterase identified by the cyclopropane carbonyl in the infrared half cyanines class fluorescence probe, the material HCY after being contacted；
The value added of the fluorescence intensity that material HCY after detection contact is sent out under excitation light at any time, the fluorescence are strong
The value added of degree at any time indicates the activity of butyrylcholine esterase in sample to be tested；The wavelength of the exciting light is 450-
750nm, the launch wavelength of the fluorescence is 680-800nm.
5th aspect, the present invention provide a kind of detection kit of the activity of BuChE, which includes：
Half cyanines class fluorescence probe of near-infrared as described above, butyrylcholine esterase standard items and reaction buffer.
The half cyanines class fluorescence probe of near-infrared of the present invention is when for detecting the activity of BuChE, with BuCh ester
Enzyme has higher affinity, the validity higher of detection.When for detecting the activity of butyrylcholine esterase, and using polypeptide
Class fluorescence probe is compared, and has higher sensitivity and selectivity；And synthetic method is simple and quick, fluorescence background value is low.Cause
This, half cyanines class probe compound fluorescence probe of near-infrared provided by the invention is for related with the activity of BuChE detection
In application, high sensitivity, good, fluorescence intensity the variation of selectivity are big, it is easy to detect, and the half cyanines class of near-infrared of the present invention is visited
Needle compound be non-peptides substrate type fluorescence probe, non-peptides fluorescence probe compared to peptides fluorescence probe stability higher,
Using it is more convenient, price is less expensive.Substrate type fluorescence probe is lower compared to suppressive fluorescence probe background values, to cytotoxicity
It is lower.
Other features and advantages of the present invention will be described in detail in subsequent specific embodiment part.
Description of the drawings
Attached drawing is to be used to provide further understanding of the present invention, and a part for constitution instruction, with following tool
Body embodiment is used to explain the present invention, but be not construed as limiting the invention together.In the accompanying drawings：
It is front and rear in significant wave with butyrylcholine esterase effect that Fig. 1 reflects the near infrared fluorescent probe of structure shown in formula (1)
Fluorescence intensity change under section.
Fig. 2 is specificity schematic diagram of the half cyanines class fluorescence probe of near-infrared to butyrylcholine esterase of structure shown in formula (1).
Specific embodiment
The specific embodiment of the present invention is described in detail below.It is it should be understood that described herein specific
Embodiment is merely to illustrate and explain the present invention, and is not intended to restrict the invention.
The endpoint of disclosed range and any value are not limited to the accurate range or value herein, these ranges or
Value should be understood to comprising the value close to these ranges or value.For numberical range, between the endpoint value of each range, respectively
It between the endpoint value of a range and individual point value and can be individually combined with each other between point value and obtain one or more
New numberical range, these numberical ranges should be considered as specific open herein.
On the one hand, the present invention provides a kind of half cyanines class fluorescence probe of near-infrared, the half cyanines class fluorescence probe of near-infrared tools
There is the structure shown in formula (1)；
Wherein, in the half cyanines class fluorescence probe of near-infrared shown in formula (1), using cyclopropane carbonyl as butyrylcholine esterase
Recognition group.
Second aspect, the preparation method the present invention also provides half cyanines class fluorescence probe of near-infrared as described above include：
Wherein, in the presence of necleophilic reaction condition and acid binding agent, in organic solvent, by structural compounds and cyclopropyl shown in formula (2)
Formyl chloride contacts；
In the preparation method of half cyanines class fluorescence probe of near-infrared of the present invention, the necleophilic reaction is in formula (2)
Hydroxyl reacted with what Cyclopropyl carbonyl chloride occurred, it is subzero 5 DEG C to 30 DEG C that the condition of the necleophilic reaction, which includes temperature, the time
For 10-200min, under preferable case, it is 0-25 DEG C that the condition of the necleophilic reaction, which is temperature, time 80-160min.
In the preparation method of half cyanines class fluorescence probe of near-infrared of the present invention, the contact needs to exist in acid binding agent
Under conditions of carry out, the acid binding agent can be any acid that can be generated in absorbing reaction commonly used in the art, avoid contact with
The acid generated in reaction influences the substance of reaction or reaction balance, it is preferable that the acid binding agent is selected from triethylamine, diethylamine, two
Isopropylamine, pyridine, N, N- dimethylamino naphthyridines, 2,6- lutidines, potassium carbonate, cesium carbonate, sodium carbonate and sodium bicarbonate
At least one of, most preferably, the acid binding agent is triethylamine.
In order to improve haptoreaction mass transfer and heat transfer effect, haptoreaction of the present invention in organic solvent environment into
Row, the organic solvent can be that any reactant that can be not with the present invention under contact conditions of the present invention reacts
And the organic solvent of suitable solution environmental can be provided for reaction, the boiling point of the organic solvent is preferably 30-150 DEG C, more
Preferably, the organic solvent for dichloromethane, tetrahydrofuran, chloroform, carbon tetrachloride, acetonitrile, acetone, glycol dimethyl ether,
At least one of n,N-Dimethylformamide and 1,2- dichloroethanes, most preferably, the organic solvent is dichloromethane.
In the preparation method of the half cyanines class fluorescence probe of near-infrared of the present invention, tied relative to 1 mM of formula (2) Suo Shi
Structure compound, the dosage of institute's Cyclopropyl carbonyl chloride is 1-2 mMs；The dosage of the acid binding agent is not particularly limited, in order to
The assimilation effect of the acid to reaction generation reached, the dosage of the acid binding agent can be 1-3 mMs, under preferable case,
The dosage of the acid binding agent is 2-3 mMs；Dosage for the organic solvent of the present invention is also not particularly limited, preferably feelings
Under condition, the dosage of the organic solvent is 20-30 milliliters.
In the preparation method of half cyanines class fluorescence probe of near-infrared of the present invention, thin-layer chromatography (TLC) can be used right
The carry out situation of reaction is monitored, and when testing result shows that raw material point disappears, shows that reaction terminates.
The preparation method of the present invention, which further includes, purifies catalytic product, and the process of purification can be included in 0-
Reaction system is washed with 20-60ml water and 20-60ml saturated common salt aqueous solutions successively at a temperature of 25 DEG C, stratification
Separation organic phase is simultaneously dried organic phase with anhydrous sodium sulfate, and removing solvent by decompression method obtains crude product, finally
Chromatography is carried out to crude product with silicagel column and obtains product.
The third aspect, the present invention also provides half cyanines class fluorescence probe of near-infrared as described above in detection BuCh ester
Application in the activity of enzyme.
The application can be any application for being related to the activity of BuChE detection in this field, for example, the application
It can include but is not limited to：The detection of the activity of BuChE, butyrylcholinesterase inhibitor screening, butyrylcholine esterase promote
It is screened into agent, Kinetic Characterization of butyrylcholinesterase inhibitor or accelerating agent etc..Application of the present invention simultaneously also can be used
In the diagnose and treat of the disease related with the activity of BuChE detection, but the present invention is not limited to this.
Preferably, the present invention provides half cyanines class fluorescence probes of near-infrared as described above to prepare for detecting butyryl courage
Application in the product of the activity of alkali esterase.
Based on application as above, fourth aspect, the present invention also provides a kind of detection method of the activity of BuChE,
This method includes：
Sample to be tested containing butyrylcholine esterase is contacted with half cyanines class fluorescence probe of near-infrared as described above so that
Butyrylcholine esterase in sample to be tested is identified by the cyclopropane carbonyl in the infrared half cyanines class fluorescence probe, is contacted
Material HCY afterwards；
The value added of the fluorescence intensity that material HCY after detection contact is sent out under excitation light at any time, the fluorescence are strong
The value added of degree at any time indicates the activity of butyrylcholine esterase in sample to be tested；The wavelength of the exciting light is 450-
750nm, the launch wavelength of the fluorescence is 680-800nm.
In the present invention, term " value added at any time " refers in the range of minute, the value added of fluorescence intensity with
The ratio of time, that is, fluorescence intensity advancing the speed at any time.
In the detection method of the activity of BuChE of the present invention, the dosage of half cyanines class fluorescence probe of near-infrared
Range is related with the concentration of butyrylcholine esterase sample to be measured, it is generally the case that the dosage energy of half cyanines class fluorescence probe of near-infrared
Enough reach the activity of characterization butyrylcholine esterase sample to be tested, under preferable case, relative to 1 μ g/mL every in sample to be tested
Butyrylcholine esterase, the dosage of the half cyanines class fluorescence probe of near-infrared can be 0.05-100 μM, best in order to reach
Detection result, the dosage of the half cyanines class fluorescence probe of near-infrared is preferably 2-10 μM.
In the detection method of the activity of BuChE provided by the invention, the butyrylcholine esterase sample to be tested with
The condition of half cyanines class fluorescence probe of near-infrared contact includes：Temperature is 25-37 DEG C, pH value 5-9, time 10-30min.It is excellent
In the case of choosing, the condition of the contact includes：Temperature is 25-30 DEG C, pH value 7-8, time 10-25min.
In detection method of the present invention, butyrylcholine esterase sample to be measured and probe (half cyanines class fluorescence of near-infrared
Probe) mixing is contacted preferably under the conditions of being protected from light, and before contact mixes, preferably to mixing buffer solution and container used
Preheated, reaction vessel can be black plate, cuvette or ELISA Plate, the preferred cuvette of reaction vessel.Preheating to be reached
Temperature be preferably 25-37 DEG C.
The condition of the detection method of the activity of BuChE of the present invention is additionally included in 5-100mM phosphate buffers
(pH=7.4) it is carried out in.
The fluorescence of material after being contacted to the butyrylcholine esterase sample to be tested with half cyanines class fluorescence probe of near-infrared is strong
The detection of degree value added at any time can carry out in microplate reader or sepectrophotofluorometer, can be used measure sample to be tested with
Fluorescence intensity changes with time value after the mixing of half cyanines class probe compound of near-infrared, to obtain the enhancement value.Furthermore, it is possible to
Ensure the reliability of detection in a manner that several control groups are tested and set in multiplicating.
When the half cyanines class fluorescence probe of near-infrared is structure shown in formula (1), method of the present invention can be used for
The determination of activity of special butyrylcholine esterase, wherein, when in sample to be tested contain elastoser, trypsase, pancreas curdled milk
During the interference of protease and carboxypeptidase, the probe molecule shown in formula (1) provided by the present invention unaffected can be used to measure
The activity of the butyrylcholine esterase of the known concentration contained in sample；Especially when containing acetylcholine ester in sample to be tested simultaneously
When enzyme and butyrylcholine esterase, the differentiation and detection of isodynamic enzyme can be carried out with formula (1) compound；By to fourth in sample to be tested
The kinetic test of acetylcholinesterase can obtain the Michaelis constant of compound hydrolysis shown in butyrylcholine esterase catalysis type (1),
So as to the activity level of butyrylcholine esterase contained in judgement sample；In addition, the chemical combination shown in formula (1) provided by the invention
Object can also be applied to detect the content of the butyrylcholine esterase contained in sample simultaneously, so as to containing butyryl in sample
The activity level of cholinesterase carries out quantitative analysis.
When the detection method of the activity of BuChE of the present invention is used for butyrylcholinesterase inhibitor or promotion
During the screening of agent, can carry out by the following method, respectively by certain density butyrylcholine esterase, certain density inhibitor or
Accelerating agent is dissolved in above-mentioned buffer solution, is placed in being preheated to preference temperature (25-37 DEG C) in Water Tank with Temp.-controlled；Reaction is held simultaneously
Device, which is placed in incubator, is preheated to preference temperature (25-37 DEG C).Then, by suitable buffering having been warmed up in reaction vessel
Solution, a certain amount of butyrylcholinesterase inhibitor or accelerating agent and suitable half cyanines class fluorescence probe of near-infrared as described above
Mixing.Butyrylcholine esterase is added under the conditions of being protected from light and starts reaction, and glimmering to sample with microplate reader or sepectrophotofluorometer
The situation of change of luminous intensity is monitored.
5th aspect, the present invention also provides a kind of detection kit of the activity of BuChE, the detection kits
Including half cyanines class fluorescence probe of near-infrared as described above, butyrylcholine esterase standard items and reaction buffer.Wherein, it is described
Butyrylcholine esterase standard items may serve as control group or draw standard curve when detecting.The reaction buffer can be
Buffer solution as described above.
Kit provided by the present invention can be applied to the activity of butyrylcholine esterase in detection sample to be tested (including sieve
Butyrylcholinesterase inhibitor or accelerating agent and the detection of other and the activity of BuChE or butyrylcholine esterase is selected to inhibit
Agent or the relevant field of accelerator function).In addition, this kit can also be used for the doctor related with the activity of BuChE detection
Detection and therapy field are learned, but the present invention is not limited to this.
In kit provided by the present invention, reaction needs to carry out in reaction buffer, it is preferable that reaction buffer is
5-100mM phosphate buffers (pH=7.4), the preservation condition of this kit is is kept in dark place at subzero 20 DEG C to 4 DEG C.The examination
The specifically used method of agent box can be carried out according to the method for act set forth below：
(1) DMSO (dimethyl sulfoxide (DMSO)) solution of half cyanines class fluorescence probe of configuration 20mM near-infrareds, and at subzero 20 DEG C
Or be kept in dark place at 4 DEG C, when use, carries out gradient dilution.
(2) the butyrylcholine esterase standard items phosphate buffer (pH=7.0) of 10mM is dissolved, is configured to 100U/ml
Butyrylcholine esterase standard solution, gradient dilution is carried out with 10 times of dilution in subzero 20 DEG C or so storages, use.
(3) phosphate buffer (pH=7.0) of the 10mM of 100 μ l of the sample to be tested containing butyrylcholine esterase is buffered
Liquid dissolves.
(4) respectively by the phosphate buffer (pH=7.0) of the 10mM of 0.2ml, half cyanines class fluorescence probe of near-infrared, to be measured
Butyrylcholine esterase sample and butyrylcholine esterase standard items are incorporated in black ELISA Plate.Utilize butyryl courage of the present invention
The activity of butyrylcholine esterase in the detection method detection sample to be tested and butyrylcholine esterase standard items of alkali esterase.
(5) standard song is drawn with the fluorescence intensity change value of the butyrylcholine esterase standard items of gradient dilution detected
Line determines the activity of butyrylcholine esterase in detected sample by comparing.
It, can be by the inhibition when the kit of the present invention is used for the screening of butyrylcholinesterase inhibitor or accelerating agent
Agent is before detection in appropriate proportions or viscosity is made an addition in reaction mixture.Then add in suitable butyrylcholine esterase standard items
Start reaction, the condition of reaction is 25-37 DEG C, pH value 5-9, time 10-30min.
The present invention will be described in detail by way of examples below.Iodate thioacetyl courage used in following embodiment
The commercially available product that it is A5751-1G purchased from Sigma-alderich Reagent Companies product identification that alkali, which is,.Soybean trypsin inhibitor is purchased
It is 10063 from Mayan company's numbering.Microplate reader is purchased from Molecular Devices companies, model SpectraMax M5.
The source of enzyme used in following embodiment and parameter are as follows：
Acetylcholinesterase is purchased from a-Aldrich (C1682) Rate activity >=1,500U/mg；
Butyrylcholine esterase is purchased from Sigma-Aldrich (C1057) Rate activity >=900U/mg；
Bovine serum albumin is 98% purchased from lark prestige scientific ＆ technical corporation (109636) purity；
Trypsase is purchased from Mayan company (10020) Rate activity >=250U/mg；
Human neutrophil elastase is purchased from Mayan company (E8140) Rate activity >=50U/mg；
Carboxypeptidase A is purchased from Worthington (CLS005304) Rate activity >=170U/mg；
Protaminase is purchased from Sigma-Aldrich Rate activities >=50U/mg；
Elastoser is purchased from Mayan company Rate activity >=30U/mg；
Chymotrypsin is purchased from Mayan company Rate activity >=1500U/mg.
(Δ F/ Δ t) are calculated fluorescence intensity change rate according to the following formula in following embodiment：
Wherein, F2Represent t2The fluorescence intensity of moment sample, F1Represent t1The fluorescence intensity of moment sample.
(Δ A/ Δ t) are calculated the change rate of ultravioletvisible absorption value according to the following formula：
Wherein, A2Represent t2The ultravioletvisible absorption value of moment sample, A1Represent t1Moment sample is ultraviolet at 412nm
Visible absorbance value.
Preparation example 1
The present embodiment is used for the preparation method for illustrating half cyanines class fluorescence probe of near-infrared of the present invention.
It, will be formula (2) Suo Shi in the presence of acid binding agent (triethylamine, 2mmol) in organic solvent (dichloromethane, 20mL)
Compound (1mmol) and Cyclopropyl carbonyl chloride (2mmol) at 5 DEG C, maintain the contact of 120min, the object after being contacted
Material.Then the material after washing contact with water (50mL) and saturated common salt aqueous solution (50mL) successively, the material after being washed.
The organic phase in material after separating, washing is simultaneously dried with anhydrous sodium sulfate, and decompression removal solvent obtains crude product, will be thick
Product obtains white solid product 0.27g through silica gel column purification.
1H NMR(400MHz,DMSO-d6):δ 8.95 (d, J=4.0Hz, 1H), 8.02 (s, 1H), 7.87 (s, 1H), 7.42
(d, J=8.0Hz, 2H), 7.25 (t, J=6.0Hz, 1H), 6.81 (s, 1H), 6.53 (s, 1H), 6.43 (d, J=8.0Hz,
1H), 5.12 (d, J=8.0Hz, 1H), 4.01 (q, J=6.0Hz, 2H), 2.73 (m, 4H), 1.41 (s, 1H), 1.25 (m,
11H),1.12(m,4H).HRMS calcd for[C31H32NO3]+:466.5841.Found:466.5425.。
Confirm that the white solid is structure shown in formula (1) with NMR and HRMS detection characterizations.
Test case 1
This test case is used to illustrate that compound and butyrylcholine esterase effect shown in the formula (1) of the offer of the present invention to be front and rear
Fluorescence intensity change.
1mg butyrylcholine esterase samples are dissolved in the butyryl of 10mM phosphate buffers (pH=7.0) preparation 1mg/L concentration
The butyrylcholine esterase standard solution that acquisition is configured is placed in 30 DEG C of Water Tanks with Temp.-controlled and preheats by cholinesterase standard solution；
96 orifice plate of black flat-bottom is placed in 30 DEG C of incubators simultaneously and is preheated.Then, the butyrylcholine esterase standard that 40 μ L is taken to be preheated
Product solution adds 120 μ L 10mM phosphate buffers (pH=7.4), and set feminine gender in 96 orifice plate of black flat-bottom of preheating
The control group buffer solution of 160 μ l (only plus), under the conditions of being protected from light, structure shown in formula (1) prepared by 40 μ L, 10 μM of preparation examples 1
Infrared half cyanines class fluorescence probe respectively with butyrylcholine esterase standard items and control group Buffer fluid contacts 5min.With microplate reader pair
Fluorescence intensity progress dynamics is swept after the contact of butyrylcholine esterase standard solution and control group solution under 665nm exciting lights
It retouches, obtains the fluorescence intensity change front and rear with butyrylcholine esterase effect of compound shown in formula (1), the result is shown in Figure 1.
As seen from Figure 1, the infrared half cyanines class fluorescence probe of structure shown in formula (1) provided by the invention is substrate fourth
There is the launch wavelength of large change, thus, it is easy to detect after acetylcholinesterase effect.
Embodiment 1
The present embodiment is used to illustrate that compound shown in formula provided by the invention (1) is detected the activity of BuChE
Method.
1mg butyrylcholine esterase samples are dissolved in 10mM phosphate buffers (pH=7.0) and carry out gradient dilution, are pressed
The concentration provided in table 1 prepares the butyrylcholine esterase standard solution of various concentration, the butyrylcholine esterase of acquisition will be configured
Standard solution is placed in 30 DEG C of Water Tanks with Temp.-controlled and preheats；96 orifice plate of black flat-bottom is placed in 30 DEG C of incubators simultaneously and is preheated.With
Afterwards, the butyrylcholine esterase standard solution that 40 μ L are preheated respectively is taken to add 120 μ L in 96 orifice plate of black flat-bottom of preheating
10mM phosphate buffers (pH=7.4), and negative control group (only plus buffer solution of 160 μ l) is set, under the conditions of being protected from light, by 40
The infrared half cyanines class fluorescence probe of structure shown in formula (1) prepared by 10 μM of preparation examples 1 of μ L respectively with butyrylcholine esterase standard items
And control group Buffer fluid contacts.Each butyrylcholine esterase standard solution and control group solution are swashed in 665nm with microplate reader
Fluorescence intensity carries out kinetic scans at 705nm after contact under shining, obtains the change of the fluorescence intensity in 5-10 minutes
As a result rate is listed in table 1.
Comparative example 1
The comparative example is used for the detection method for illustrating the activity of BuChE in the prior art.
Using the substrate water of method detection butyrylcholine esterase standard items catalysis series concentration gradient same as Example 1
The situation of solution, unlike, substrate probe used is acetylthiocholine iodide (purchased from Sigma-Aldrich companies, product
Number is A5751-1G), and after excessive DTNB (50 μM, 5,5'- bis- thiobis (2- nitrobenzoic acids)) is added in, in wavelength
For docking at 412nm, ultravioletvisible absorption value carries out kinetic scans after touch, obtains the ultravioletvisible absorption in 5-10 minutes
As a result the change rate of value is listed in table 1.
Table 1
The method provided using this law can be seen that by the result of embodiment 1, it can be to the elastin laminin in sample
The activity of enzyme carries out the detection of simple and sensitive, and can be seen that by the result of embodiment 1 and comparative example 1 and pass through the present invention
The method of offer, formula (1) compound represented fluorescence probe and existing iodate used in method provided by the present invention
Acetylthiocholine probe, which is compared, has higher sensitivity and affinity.
Embodiment 2
The present embodiment is used to illustrate the infrared half cyanines class fluorescence probe pair of non-peptides shown in using formula provided by the invention (1)
The method that the activity of BuChE is measured in sample containing butyrylcholine esterase.
Respectively by butyrylcholine esterase and containing butyrylcholine esterase sample (be respectively contain butyrylcholine esterase
Elastin laminin enzyme sample, trypsin sample, protaminase sample and chymotrypsin-like product, wherein, butyrylcholine esterase
Weight ratio with other each enzymes is 1:199) it is dissolved in 10mM phosphate buffers (pH=7.4) buffer solution and is diluted, match
Into the enzyme solutions (total enzyme concentration) of a concentration of 10mg/L；96 orifice plate of black flat-bottom is placed in 30 DEG C of incubators simultaneously and is preheated.With
Afterwards, the enzyme solutions that 40 μ L are preheated respectively are taken in 96 orifice plate of black flat-bottom of preheating, add 120 μ L 10mM phosphate buffers
(pH=7.4) buffer solution, and negative control group (only plus buffer solution of 160 μ l) is set, under the conditions of being protected from light, by 40 μ L formulas (1)
The probe of shown structure contacts respectively with enzyme solutions and control group, is existed with microplate reader to enzyme standard solution and control group solution
Fluorescence intensity carries out kinetic scans at 705nm after contact under the exciting light of 665nm, obtains the fluorescence in 5-10 minutes
As a result the change rate of intensity is listed in table 2.
Comparative example 2
This comparative example is used to illustrate using existing acetylthiocholine iodide probe to the sample containing butyrylcholine esterase
The method that the activity of BuChE is measured in product.
Using the sample of method same as Example 2 to butyrylcholine esterase and containing butyrylcholine esterase (respectively
For the elastin laminin enzyme sample containing butyrylcholine esterase, trypsin sample, protaminase sample and chymotrypsin-like
Product) in the activity of BuChE be measured, unlike, used substrate probe for acetylthiocholine iodide (purchase
From Sigma-Aldrich companies, product identification A5751-1G), and after excessive DTNB (50 μM) is added in, be in wavelength
Ultravioletvisible absorption value carries out kinetic scans after touch for docking at 412nm, obtains the ultravioletvisible absorption value in 5-10 minutes
Change rate, be as a result listed in table 2.
Table 2
It can be seen that compared with prior art by the result of embodiment 2 and comparative example 2, method provided by the present invention
In can carry out effective, quick and easy active testing to the butyrylcholine esterase in a variety of sources, and with prior art institute
The method used is compared, and the fluorescence intensity change of method provided by the present invention becomes apparent from, and the sensitivity of detection and affinity are more
It is high.
Test case 2
This test case is used for the specificity for illustrating the activity of BuChE detection method provided by the invention.
Respectively by 1mg samples to be tested (acetylcholinesterase, elastoser, trypsase, chymotrypsin, carboxylic peptide
Enzyme A, protaminase, bovine serum albumin(BSA), butyrylcholine esterase) it is dissolved in 10mM phosphate buffers (pH=7.0) buffer solution and matches
The solution for putting the sample to be tested of a concentration of 100mg/L is placed in preheating in 30 DEG C of Water Tanks with Temp.-controlled；Simultaneously by 96 orifice plate of black flat-bottom
It is placed in 30 DEG C of incubators and preheats.Then, the solution of sample to be tested that 40 μ L are preheated respectively is taken in 96 hole of black flat-bottom of preheating
In plate, 120 μ L 10mM phosphate buffers (pH=7.4) buffer solutions are added, under the conditions of being protected from light, will be tied 40 μ L formulas (1) Suo Shi
The half cyanines class fluorescence probe of near-infrared of structure contacts respectively with each sample to be tested, while sets the control group for not adding sample to be tested,
Various samples to be tested in 10 minutes are detected under the exciting light of 665nm be catalyzed compound hydrolysis shown in 40 μ L formulas (1) with microplate reader
Relative speed, the results are shown in Figure 2.
Butyrylcholine esterase can be seen that used in method provided by the present invention by the result of test case 2
The hydrolysis ability of half cyanines class fluorescence probe of near-infrared shown in formula (1) is especially significantly high significantly higher than other kinds of enzyme
In isodynamic enzyme acetylcholinesterase.Therefore, half cyanines class fluorescence probe is very high to the specificity of enzyme used in method of the invention,
It can realize the specific detection of butyrylcholine esterase.
Embodiment 3
The present embodiment is used for the screening technique for illustrating butyrylcholinesterase inhibitor according to the present invention.
1mg butyrylcholine esterase samples are dissolved in configuration concentration in 10mM phosphoric acid acid buffer (pH=7.0) buffer solution
For the butyrylcholine esterase standard solution to be measured of 1.5mg/mL, inhibitor Tacrine (tacrine) is dissolved in 1mL 10mM
Be configured in phosphoric acid acid buffer (pH=7.0) buffer solution concentration gradient for 0.004,0.008,0.016,0.032,0.04,
The fluorescence probe of structure shown in 20mmol formulas (1) is dissolved in 2mL 10mM phosphoric acid acid buffers by 0.08 μM of inhibitor solution
(pH=7.0) the fluorescence probe solution of a concentration of 0.2mM is configured in buffer solution, the butyrylcholine esterase obtained will be configured respectively
Standard solution, Tacrine and fluorescence probe solution are placed in 30 DEG C of Water Tanks with Temp.-controlled and preheat；96 orifice plate of black flat-bottom is put simultaneously
It is preheated in 30 DEG C of incubators.Then, respectively by the Tacrine solution of 50 each concentration gradients of μ L, 90 μ L 10mM phosphoric acid acid bufferings
Liquid (pH=7.4) buffer solution and 10 μ l fluorescence probes (final concentration of 10 μM) solution are added in 96 orifice plate of black flat-bottom and are mixed into
Experimental group, and set up 140 μ L 10mM phosphoric acid acid buffer (pH=7.4) buffer solutions and pair of 10 μ L fluorescence probes solution mixing
According to group, under the conditions of being protected from light, 50 μ L butyrylcholine esterase standard items are rapidly added with the volley of rifle fire in experimental group and control group respectively
Solution starts reaction.With microplate reader to fluorescence intensity carries out at 705nm after contact of each group solution under the exciting light of 665nm
Kinetic scans obtain the change rate of the fluorescence intensity in 5-10 minutes, are as a result listed in table 3.
Comparative example 3
This comparative example is for explanation using the screening technique of the butyrylcholinesterase inhibitor of the prior art.
The screening of butyrylcholinesterase inhibitor is carried out using method same as Example 3, unlike, fluorescence is visited
Needle solution is changed to the acetylthiocholine iodide (CAS of Sigma-Aldrich Reagent Companies:1866-15-5), it is and excessive adding in
DTNB reagents (50 μM) after, in wavelength, for the docking of 412nm places, ultravioletvisible absorption value carries out kinetic scans, acquisition the after touch
The change rate of ultravioletvisible absorption value in 5-10 minutes, is as a result listed in table 3.
Table 3
It can be seen that method provided by the present invention can be used in BuCh by the result of embodiment 3 and comparative example 3
The screening of esterase inhibitor.Also, it is noted that the screening technique of butyrylcholinesterase inhibitor provided by the invention with
It is compared using acetylthiocholine iodide probe, the larger progress for being easy to detection of the variation range of fluorescence signal.Therefore, it is of the invention
The direct detecting method provided has higher sensitivity.
In addition, for the accuracy of method, half cyanines class fluorescence probe of near-infrared provided by the present invention is BuCh
The substrate type probe of esterase can directly be reflected the activity of enzyme after enzyme hydrolysis；And the Ellman side used in comparative example 1-3
Method (acetylthiocholine iodide provided using Sigma-Aldrich Reagent Companies makees substrate) then needs to add in coupling reagent
(DTNB) determination of activity of enzyme can just be carried out.Therefore, half cyanines class fluorescence probe provided by the present invention and its to BuCh
The detection of esterase is more directly and accurate.
The preferred embodiment of the present invention has been described above in detail, still, during present invention is not limited to the embodiments described above
Detail, within the scope of the technical concept of the present invention, a variety of simple variants can be carried out to technical scheme of the present invention, this
A little simple variants all belong to the scope of protection of the present invention.
It is further to note that specific technical features described in the above specific embodiments, in not lance
In the case of shield, can be combined by any suitable means, in order to avoid unnecessary repetition, the present invention to it is various can
The combination of energy no longer separately illustrates.
In addition, various embodiments of the present invention can be combined randomly, as long as it is without prejudice to originally
The thought of invention, it should also be regarded as the disclosure of the present invention.
Claims (10)
1. a kind of half cyanines class fluorescence probe of near-infrared, which is characterized in that the half cyanines class fluorescence probe of near-infrared has formula (1) institute
The structure shown：
2. the preparation method of half cyanines class fluorescence probe of near-infrared described in claim 1, which is characterized in that this method is included in parent
In the presence of nuclear reaction condition and acid binding agent, in organic solvent, formula (2) compound represented is contacted with Cyclopropyl carbonyl chloride；
3. preparation method according to claim 2, wherein, relative to structural compounds, institute shown in 1 mM of formula (2)
The dosage for stating Cyclopropyl carbonyl chloride is 1-2 mMs, and the dosage of the acid binding agent is 1-3 mMs, the use of the organic solvent
Measure is 20-30 milliliters.
4. the preparation method according to Claims 2 or 3, wherein, the acid binding agent is selected from triethylamine, diethylamine, diisopropyl
Base amine, pyridine, N, in N- dimethylamino naphthyridines, 2,6- lutidines, potassium carbonate, cesium carbonate, sodium carbonate and sodium bicarbonate
It is at least one；The organic solvent is selected from dichloromethane, tetrahydrofuran, chloroform, carbon tetrachloride, acetonitrile, acetone, glycol dinitrate
At least one of ether, N,N-dimethylformamide and 1,2- dichloroethanes.
5. according to the preparation method described in any one in claim 2-4, wherein, the necleophilic reaction condition includes：Temperature
It is subzero 5 DEG C to 30 DEG C, time 10-120min.
6. application of the half cyanines class fluorescence probe of near-infrared described in claim 1 in the activity of detection butyrylcholine esterase.
7. a kind of detection method of the activity of BuChE, which is characterized in that this method includes：
Sample to be tested is contacted with the half cyanines class fluorescence probe of near-infrared described in claim 1 so that the fourth in sample to be tested
Acetylcholinesterase identified by the cyclopropane carbonyl in the infrared half cyanines class fluorescence probe, the material HCY after being contacted；
The value added of the fluorescence intensities that send out under excitation light of material HCY at any time after detection contact, the fluorescence intensity with
The value added of time indicates the activity of butyrylcholine esterase in sample to be tested；The wavelength of the exciting light be 450-750nm, institute
The launch wavelength for stating fluorescence is 680-800nm.
8. the detection method described in claim 7, wherein, relative to the butyrylcholine esterase of 1 μ g/mL, half cyanines of near-infrared
The dosage of class fluorescence probe is 0.05-100 μM.
9. the detection method described in claim 7 or 8, wherein, the condition of contact includes：Temperature is 25-37 DEG C, pH value 5-9,
Time is 10-30min.
10. a kind of detection kit of the activity of BuChE, the detection kit include：It is described in claim 1 near red
Outer half cyanines class fluorescence probe, butyrylcholine esterase standard items and reaction buffer.
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| CN110129033A (en) * | 2018-08-06 | 2019-08-16 | 浙江师范大学 | A kind of fluorescence probe and its preparation method and application detecting the activity of BuChE |
| CN111592504A (en) * | 2020-06-12 | 2020-08-28 | 青岛科技大学 | Fluorescent probe for detecting butyrylcholine esterase activity and synthetic method and application thereof |
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| CN109929548B (en) * | 2019-04-15 | 2020-10-09 | 济南大学 | Novel near-infrared fluorescent probe for detecting carboxypeptidase A |
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